Group A streptococci (GAS) are highly specific human pathogens. Their efficient colonization and dissemination within the host lead to a spectrum of local and invasive infections, as well as infectious sequel, e.g., rheumatic heart disease, streptococcal toxic shock syndrome, and necrotic fasciitis. GAS surface M and M-like proteins are critical virulence factors of these organisms, and function through various mechanisms to allow effective infectivity in the host. One important such mechanism depends on the activation of the host fibrinolytic system by these bacteria, and M (e.g., M1) and M-like (e.g., PAM) proteins are essential for this function in large part through their ability to bind to host human plasminogen (hPg) directly (PAM), or, indirectly (M1), via fibrinogen (Fg)/fibrin (Fn). Through this overal mechanism, M and M-like proteins enhance the activation of Pg to the serine protease, plasmin (Pm), by bacterial or host Pg activators, thus creating a proteolytic bacterial microenvironment employed by GAS to catalyze degradation of fibrin, the extracellular matrix, and/or connective tissue, which then assists the spread of the bacteria. We will employ structural biochemistry, as well as bacterial and mammalian genetics, to understand the in vitro and in vivo mechanistic relationships between GAS proteins that mediate its virulence via the host fibrinolytic system, and their reliance on specific proteins of this system. Specifically, we propose three aims: 1. To study the relationships between structural domains within a M-like protein (PAM, from GAS strain AP53) and an M protein (M1, from GAS strain SF370) in their in vitro functioning using structural biochemical tools. Emphasis will be placed on establishing the correspondence between 1o and 2o structures in these proteins with hPg and/or Fg binding and activation of hPg by bacterial streptokinase (SK) and host activators. 2. To design and study variant AP53 strains with altered expression of PAM and SK in order to change the mechanism of assimilation of Pm on GAS, or to render GAS deficient in its capacity to activate hPg. The virulence of these mutated GAS strains, in a murine invasive skin infection model, will be examined in mice with investigator imposed genetic alterations in the fibrinolytic system. 3. To employ a continual venous catheterization infusion method to administer hPg and mutants of hPg into Pg-/- mice to examine the roles of individual domains of hPg in its activation to Pm in GAS, and the roles of these variant Pgs in promoting AP53 virulence. The overall goal of this work is to mechanistically assess interactions of the bacteria and the host in activation of the fibrinolytic system, and their relationships to GAS pathogenicity. This will allow the establishment of new paradigms for treatment of diseases associated with these infections. PUBLIC HEALTH RELEVANCE: Group A streptococcal (GAS) infections affect >700M people annually worldwide, with ~18M of these considered severe, and another ~600K of invasive types with a fatality rate of ~30%. There are more than 200 strains of GAS that have been identified. Among the important GAS virulence mechanisms is their ability to subvert the host fibrinolytic system to benefit the dissemination of the bacteria by dissolving the fibrin matrix that encapsulates the bacteria. Also, activation of the fibrinolytic system potentially results in a cascade of matrix metalloproteinase activations, thus assisting bacterial metastasis into deep tissues, causing life-threatening infections. We will study the mechanisms by which this occurs to potentially allow new strategies to be used to attenuate this group of morbid and oftentimes fatal diseases.